For decades, the Drosophila larval neuromuscular junction (NMJ) has been a powerful model system for genetic and molecular dissection of synaptic growth, structure, and function. More recently the peripheral nervous system of third instar larvae has been employed to study acute neuronal responses to axon damage and disease. However, due to the short time interval between the third larval instar and pupariation, the system is not well suited to study processes that extend over a longer time period. Recent studies demonstrate that third instar larvae mount a rapid initial response to axon damage and display tantalizing beginnings of axonal regrowth. However, the onset of metamorphosis with replacement of most larval tissues precludes more complete analysis of the response to injury - including involvement of glia and possible axonal repair - over time. Similarly, the window of observation in experiments probing mechanisms that maintain NMJ structure and function over time, or how these are compromised with age or by disease, is significantly limited by the onset of pupariation. The goal of this application is to characterize and demonstrate the utility of an experimental system we are developing that overcomes these time constraints while preserving the features of the larval NMJ that makes it such a powerful model. We exploit genetic variants in which larvae develop normally but subsequently remain in the third instar for up to 10 days (4 times longer than normal), during which time they continue to grow before finally undergoing metamorphosis and eclosion. On the basis of our preliminary results, we are confident that the expanded third instar lifespan provides a novel and powerful opportunity for experiments that probe time-dependent neurobiological processes. To establish the validity and utility of this Extended Larval Life-span (ELL) model, we propose experiments that aim to answer the following questions: (1) Is NMJ growth normal in ELL larvae during development? Does the NMJ continue to grow along with the increase in larval size during ELL? Do the key signaling pathways known to regulate NMJ growth during normal development continue to function during ELL? (2) Does the NMJ remain structurally and functionally intact throughout ELL? (3) Can we prove the utility of the ELL system as an experimental tool by employing it to expand our understanding of the injury response in larval motor axons and peripheral nerve glia over an extended time frame? We believe that this novel experimental system has enormous potential to greatly expand the power of the larval NMJ as a model system and enable us to make unique inroads in studies of axonal regeneration and synaptic maintenance, both of which are highly relevant for understanding and treatment of a number of human neurological disorders. PUBLIC HEALTH RELEVANCE: Many human neurological disorders are associated with impairment of motor pathways either as a result of physical damage to nerves caused by injury or as a result of various diseases that perturb the structure or function of synapses where nerves relay electrical signals to muscles. To fully understand these and related disorders and to develop rational therapies, we require a model system in which the necessary genetic, molecular, and cellular analyses can be carried out. We are developing a novel experimental system, long-lived Drosophila larvae, that offers numerous advantages for studies of this type and we are applying it to study the process of nerve regrowth after injury.